質(zhì)粒圖譜

Position Feature Position Feature

Start: 62 End: 352 CYC1 promoter Start: 2499 End: 4709 LEU2 auxotrophic marker

Start: 364 End: 969 LexA DNA binding domain Start: 4858 End: 5891 KanR resistance gene

Start: 988 End: 1224 VP16 transactivation domain Start: 6184 End: 6851 pBluescript origin of replication

Start: 1231 End: 1365 Cub, amino acids 34-76 of ubiquitin Start: 6973 End: 7512 CEN/ARS origin of replication

Start: 1509 End: 1768 CYC1 terminator

Multispan integral membrane proteins (i.e. those having multiple transmembrane domains)

with an Nout/Cin topology often do not have a signal sequence. We recommend that you use

the vector pBT3-STE for these proteins. The STE2 leader sequence in pBT3-STE is derived

from the Saccharomyces cerevisiae Ste2 protein (a G protein coupled receptor). It does not

have any targeting function but only serves as a short leader sequence to optimize expression

of your bait. The Cub-LexA-VP16 cassette is fused to the C-terminus of the bait.

On the other hand, if you have a protein whose C-terminus is lumenal, you should use

pBT3-N to fuse the LexA-VP16-Cub module to the cytosolic N-terminus of your protein of

interest. The vector pBT3-N is specifically designed for screening type II integral membrane

proteins.

Subclone the cDNA encoding your type II

protein into the vector pBT3-N. No leader

sequence is required. Please note that the

orientation of the module is reversed in

pBT3-N (LexA-VP16-Cub) to allow

separation of the transcription factor from

the bait via UBP cleavage.